Nucleic Acids and Libraries

Abstract

The invention relates to a nucleic acid comprising the following contiguous elements arranged in the 5 prime to 3 prime direction; a promoter; a selectable marker; a cloning site for receipt of a nucleic acid segment, said segment comprising a candidate miRNA target sequence; and a poly adenylation signal, said elements arranged such that a transcript directed by said promoter comprises said selectable marker, said candidate miRNA target sequence, and said poly adenylation signal in that order. Suitably the miRNA test sequence is or is derived from a 3′UTR. The invention also relates to methods for making and screening libraries.

1 .- 21 . (canceled) 
     
     
         22 . A method for identifying a miRNA target sequence comprising the steps of
 (a) providing a nucleic acid comprising the following contiguous elements arranged in the 5 prime to 3 prime direction;
 a) a promoter; 
 b) at least two selectable markers; 
 c) a cloning site for receipt of a nucleic acid segment, said segment comprising a candidate regulatory RNA target sequence; and 
 d) a poly adenylation signal, 
 said elements arranged such that a transcript directed by said promoter comprises said at least two selectable markers, said candidate regulatory RNA target sequence, and said poly adenylation signal in that order; and 
 further comprising a candidate miRNA target sequence; 
   (b) introducing said nucleic acid of (a) into a host cell;
 (c) selecting host cell(s) expressing at least one selectable marker of said nucleic acid; 
   (d) introducing at least one miRNA of interest to said host cell(s) of (c), and
 (e) assaying for expression of at least one selectable marker of said nucleic acid in the cells of (d), 
   wherein if the cells of (d) do not show expression of at least one selectable marker then the candidate miRNA target sequence is identified as a miRNA target sequence.   
     
     
         23 . A method for identifying an miRNA active against a miRNA target sequence comprising the steps of
 (a) providing a nucleic acid comprising the following contiguous elements arranged in the 5 prime to 3 prime direction;
 a) a promoter; 
 b) at least two selectable markers; 
 c) a cloning site for receipt of a nucleic acid segment, said segment comprising a candidate regulatory RNA target sequence; and 
 d) a poly adenylation signal, 
 said elements arranged such that a transcript directed by said promoter comprises said at least two selectable markers, said candidate regulatory RNA target sequence, and said poly adenylation signal in that order; and 
 further comprising said miRNA target sequence; 
 (b) introducing the nucleic acid of (a) into a host cell; 
 (c) selecting host cell(s) expressing at least one selectable marker of said nucleic acid; 
 (d) introducing at least one miRNA of interest to said host cell(s) of (c), and 
 (e) assaying for expression of at least one selectable marker of said nucleic acid in the cells of (d), 
   wherein if the cells of (d) do not show expression of at least one selectable marker then the miRNA of interest is identified as an miRNA active against said miRNA target sequence.   
     
     
         24 . The method according to  claim 22  wherein step (e) comprises selecting against cells which express at least one selectable marker. 
     
     
         25 . The method according to  claim 22  wherein step (e) comprises selecting for cells which do not express at least one selectable marker. 
     
     
         26 .- 31 . (canceled) 
     
     
         32 . The method according to  claim 23  wherein step (e) comprises selecting against cells which express at least one selectable marker. 
     
     
         33 . The method according to  claim 23  wherein step (e) comprises selecting for cells which do not express at least one selectable marker.